Suspensions of cyclosporin A form 2

ABSTRACT

Disclosed herein are methods of formulating cyclosporin A Form 2.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application is a continuation of copending U.S. patentapplication Ser. No. 14/325,167, filed Jul. 7, 2014, which is adivisional of U.S. patent application Ser. No. 13/677,014, filed Nov.14, 2012, now issued as U.S. Pat. No. 8,796,222, which claims priorityto U.S. Provisional Patent Application No. 61/559,866, filed Nov. 15,2011, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the fields of nanotechnology and drugformulation technology.

BACKGROUND

Cyclosporine A is the active ingredient in Restasis®, a drug that isused to treat dry eye disease. Cyclosporin A is poorly soluble in water,and so is currently formulated either by dissolving the drug in oil toform an emulsion, or by mixing the drug with high levels of surfactantsand/or solubilizers to form an aqueous solution. The inventors havediscovered a formulation of cyclosporin A using a new crystallinepolymorph of cyclosporin A, to create nanosuspensions comprisingparticles of cyclosporin A having an average size of around 1 micrometeror less (to put that number in perspective, the average thickness of ahuman hair is around 100 micrometers).

A nanosuspension of cyclosporin A, when delivered topically to the eye,may have one or more advantages, including the following:

-   -   a higher bioavailability compared to suspensions, due to the        higher surface area available for dissolution;    -   a longer retention on the eye due to smaller particles, leading        to further improvement in bioavailability;    -   a lower potential for foreign body sensation or particle        irritation, thus reducing tearing and drainage of formulations        from the eye;    -   a lower level of surfactants or solubilizers in the formulation,        improving tolerability and bioavailability of the drug.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the particle size distribution of cyclosporin A Form 2nanosuspensions prepared using a high pressure homogenizer compared tosuspensions prior to milling.

FIG. 2 compares the particle size distribution of cyclosporin A Form 2nanosuspensions prepared using different surfactants and stabilizers.

FIG. 3 shows the particle size distribution of Formulation diluted invehicle containing Na CMC.

FIG. 4 shows average particle size of a nanosuspension using cyclosporinA Form 3 compared to a nanosuspensions using Form 2 after milling usinga microfluidizer. Form 2 Forms a nanosuspension (particle size <1 μm)while Form 3 does not.

FIGS. 5-13 show the results when four different formulations accordingto the invention (summarized in Table 2) and Restasis® are administeredto NZW female rabbits (two rabbits total, one formulation per eye) in asingle topical dose:

FIG. 5 shows concentrations in the cornea.

FIG. 6 summarizes pharmacokinetic data.

FIG. 7 shows concentrations in the bulbar conjunctiva.

FIG. 8 shows concentrations in the palpebral conjunctiva.

FIG. 9 summarizes pharmacokinetic data for the bulbar conjunctiva andpalpebral conjunctiva.

FIG. 10 shows concentrations in the bulbar conjunctiva.

FIG. 11 shows concentrations in the palpebral conjunctiva.

FIG. 12 shows concentrations in the lacrimal gland.

FIG. 13 summarizes pharmacokinetic data for lacrimal gland.

FIG. 14 depicts characteristic X-ray powder diffraction (XRPD) patternsof CsA in a new crystalline form (designated as Form 2 herein),tetragonal form (designated as Form 1 herein), and orthorhombic form(designated as Form 3 herein).

FIG. 15 depicts the XRPD diffractogram of CsA crystalline Form 2.

FIG. 16 depicts the water sorption/desorption profile of CsA Form 2.

FIG. 17 depicts MDSC analysis of CsA Form 2 recovered from 0.04%formulation with 1% PS80.

FIG. 18 shows the simulated XRPD pattern of cyclosporine A forms.

DETAILED DESCRIPTION

Cyclosporin A (CsA) is a cyclic peptide having the following chemicalstructure:

Its chemical name iscyclo[[(E)-(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6-octenoyl]-L-2-aminobutyryl-N-methylglycyl-N-methyl-L-leucyI-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl].It is also known by the names cyclosporine, cyclosporine A, ciclosporin,and ciclosporin A. It is the active ingredient in Restasis® (Allergan,Inc., Irvine, California), an emulsion comprising 0.05% (w/v)cyclosporin. Restasis® is approved in the United States to increase tearproduction in patients whose tear production is presumed to besuppressed due to ocular inflammation associated withkeratoconjunctivitis sicca.

Cyclosporin A Form 2

Cyclosporin A is known to exist in an amorphous form, liquid crystalform, tetragonal crystalline form (form 1), and an orthorhombic form(form 3). A new crystalline form, cyclosporin A Form 2, has recentlybeen discovered.

The XRPD pattern of CsA Form 2 differs significantly from the tetragonalform and orthorhombic form (FIG. 14). The major crystalline peaks forCsA form 2 appear at (2θ) when scanned by an X-ray diffractometer withX-ray source as Cu Kα radiation, λ=1.54 Å, at 30 kV/15 mA: 7.5, 8.8,10.2, 11.3, 12.7, 13.8, 14.5, 15.6 and 17.5 (d-spacing in crystallattice at about 11.8, 10.0, 8.7, 7.8, 7.0, 6.4, 6.1, 5.6 and 5.1 {acuteover (Å)}, respectively, FIG. 15). These major peaks are defined asthose being unique to Form 2 relative to the orthorhombic or tetragonalforms; as well as, peaks having an intensity greater than 5 times thebackground.

In one embodiment, the new crystalline form (Form 2) of CsA is anonstoichiometric hydrate of Cyclosporin A. In another embodiment, thecrystalline Form 2 is represented by the formula:

wherein X is the number of molecules of water and varies from 0 to 3. Inone embodiment, X in the above formula is 2.

Form 2 appears to be a kinetically stable form of CsA in aqueoussuspensions. Suspensions containing Form 2 show no conversion to otherknown polymorphic or pseudomorphic forms upon storage. It has been foundthat Form 1 and the amorphous form convert to Form 2 in the presence ofwater.

The single crystal structure of the hydrate form of CsA Form 2 has beendetermined and the crystal structure parameters are listed in Table 2.These results indicate that Form 2 is unique compared to other knowncrystalline forms of cyclosporine A.

TABLE 1 Crystal data and data collection parameters of crystal structuresolution of CsA Form 2. formula C₆₂H₁₁₅N₁₁O₁₄ formula weight 1238.67space group P 2₁ 2₁ 2₁ (No. 19) a (Å) 12.6390(5) b (Å) 19.7582(8) c (Å)29.568(2) volume (Å³) 7383.8(7) Z 4 d_(calc) (g cm⁻³) 1.114 crystaldimensions (mm) 0.27 × 0.18 × 0.12 temperature (K) 150 radiation(wavelength in Å) Cu K_(a) (1.54184) monochromator confocal opticslinear abs coef (mm⁻¹) 0.640 absorption correction applied empirical^(a)transmission factors (min, max) 0.80, 0.93 diffractometer RigakuRAPID-II h, k, l range −13 to 13 −21 to 21 −32 to 21 2θ range (deg)5.38-115.00 mosaicity (deg) 1.31 programs used SHELXTL F_(coo) 2704.0weighting 1/[σ²(Fo²) + (0.0845P)² + 0.0000P] where P = (Fo² + 2Fc²)/3data collected 37360 unique data 9964 R_(int) 0.077 data used inrefinement 9964 cutoff used in R-factor calculations F_(o) ² >2.0s(F_(o) ²) data with I > 2.0s(I) 6597 number of variables 834 largestshift/esd in final cycle 0.00 R(F_(o)) 0.061 R_(w)(F_(o) ²) 0.145goodness of fit 1.037 absolute structure determination Flackparameter^(b)(0.0(3))

The asymmetric unit of this CsA Form 2 contains one cyclosporine Amolecule and two water molecules. It is possible that any small moleculethat can hydrogen bond to water could play the role of space filler,which would give a range of potential structures running from theorthorhombic dihydrate to distorted monoclinic dihydrate The XRPDpattern calculated from the single-crystal structure is shown in FIG. 8and it matches the experimental pattern shown in FIG. 2. These matchingpatterns further corroborate that Form 2 is a unique and purecrystalline form of cyclosporine A.

Without wishing to be bound by theory, thermogravimetric analysiscombined with KF titration and vapor sorption desorption analysis (VSA)suggest that CsA Form 2 is a non-stoichiometric hydrate of CsA. Thevapor sorption analysis of Cyclosporine Form 2 indicates that watercontent in the new crystal form reversibly varies with relative humidityas shown in FIG. 16. Similar to the tetragonal form, the new CsA formundergoes a phase transition to a liquid crystal or amorphous form at124.4° C. prior to melting as indicated by the modulated differentialcalorimetric (MDSC) analysis (FIG. 17).

Cyclosporin A Form 2 may be obtained by suspending amorphous 0.05%cyclosporin A (w/v) in 1% Polysorbate 80, heating the solution to 65°C., holding it at that temperature for 24 hours, and then recovering theprecipitate by vacuum filtration. One can then use the cyclosporin AForm 2 thus obtained to generate additional amounts, using Cyclosporin AForm 2 as a seed crystal; in this method, one suspends about 30 gcyclosporin A in a solution of 900 ml water containing 1% (w/v)Polysorbate 80, heats the solution to 65° C., and then seeds it with 0.2g of cyclosporin A Form 2 at a temperature of 52° C. The solution isthen stirred for about 22 hours at a temperature of between about 61° C.and 65° C., and then recovers the precipitate that results.

Further details regarding CsA Form 2 may be found in U.S. patentapplication Ser. No. 13/480,710, the entire contents of which areincorporated by reference herein.

Suspensions of Cyclosporin A Form 2

Compositions of the invention are ophthalmically acceptable suspensionsof Cyclosporin A form 2. By “ophthalmically acceptable,” the inventorsmean that the suspensions are formulated in such a way as to benon-irritating when administered to the eye of a mammal, such as ahuman. In one embodiment, the compositions are suspensions; that is,they comprise particles of cyclosporin A form 2, having an averageparticle size greater than about 1 μm, dispersed throughout a liquidvehicle. In another embodiment, the compositions are nanosuspensions;that is, they comprise particles of cyclosporin A form 2, having anaverage particle size of less than about 1 μm, that are dispersedthroughout a liquid vehicle.

In one embodiment, the suspension comprises cyclosporin A form 2 at aconcentration of about 0.001% to about 10% (w/v). In one embodiment, thesuspension comprises cyclosporin A form 2 at a concentration of about0.001% (w/v) to about 0.01%, about 0.001% (w/v) to about 0.04% (w/v),about 0.001% (w/v) to about 0.03% (w/v), about 0.001% (w/v) to about0.02% (w/v), or about 0.001% (w/v) to about 0.01% (w/v). In anotherembodiment, the suspension comprises cyclosporin A form 2 at aconcentration of about 0.01% (w/v) to about 0.05%, about 0.01% (w/v) toabout 0.04% (w/v), about 0.01% (w/v) to about 0.03% (w/v), about 0.01%(w/v) to about 0.02% (w/v), or about 0.01% (w/v) to about 0.01% (w/v).In another embodiment, the suspension comprises cyclosporin A form 2 ata concentration of about 0.01% (w/v) to about 0.1%, about 0.1% (w/v) toabout 0.5% (w/v), about 0.01% (w/v) to about 1% (w/v), or about 1% (w/v)to about 10%.

For example, the suspensions may comprise about 0.001% (w/v), about0.002% (w/v), about 0.003% (w/v), about 0.004% (w/v), about 0.005%(w/v), about 0.006% (w/v), about 0.007% (w/v), about 0.008% (w/v), about0.009% (w/v), about 0.01% (w/v), about 0.015% (w/v), about 0.02% (w/v),about 0.025% (w/v), about 0.03% (w/v), about 0.035% (w/v), about 0.04%(w/v), about 0.045% (w/v), about 0.05% (w/v), about 0.055% (w/v), about0.06% (w/v), about 0.065% (w/v), about 0.07% (w/v), about 0.075% (w/v),about 0.08% (w/v), about 0.085% (w/v), about 0.09% (w/v), about 0.095%(w/v), about 0.1% (w/v), about 0.15% (w/v), about 0.2% (w/v), about0.25% (w/v), about 0.3% (w/v), about 0.35% (w/v), about 0.4% (w/v),about 0.45% (w/v), about 0.5% (w/v), about 0.55% (w/v), about 0.6%(w/v), about 0.65% (w/v), about 0.7% (w/v), about 0.75% (w/v), about0.8% (w/v), about 0.85% (w/v), about 0.9% (w/v), about 0.95% (w/v), orabout 1.0% (w/v) cyclosporin A form 2.

In one embodiment, the suspension comprises a surfactant. In oneembodiment, the surfactant is selected from polyoxyethylene (20)sorbitan monooleate (Polysorbate 80), polyethylene glycol 660hydroxystearate (Solutol), polyoxyethylene (40) stearate Myrj 52(POE-40-Stearate), pluronic F68 (Poloxamer 188), polyoxyethylenesorbitan monolaurate (Polysorbate 20), and sodium glycocholate (NaGC).The vehicle may contain all of these surfactants, or one, two, three,four, or five of them.

One can use between about 0.001% (w/v) and about 5% (w/v) of thesurfactant. In one embodiment, the suspensions contain about 0.001%(w/v) to about 1% (w/v), about 0.001% (w/v) to about 0.1% (w/v), about0.01% (w/v) to about 0.1% (w/v), or about 0.1% (w/v) to about 1% (w/v)of the surfactant. For example, the suspensions may contain about 0.001%(w/v), about 0.002% (w/v), about 0.003% (w/v), about 0.004% (w/v), about0.005% (w/v), about 0.006% (w/v), about 0.007% (w/v), about 0.008%(w/v), about 0.009% (w/v), about 0.01% (w/v), about 0.02% (w/v), about0.03% (w/v), about 0.04% (w/v), about 0.05% (w/v), about 0.06% (w/v),about 0.07% (w/v), about 0.08% (w/v), about 0.09% (w/v), about 0.1%(w/v), about 0.2% (w/v), about 0.3% (w/v), about 0.4% (w/v), about 0.5%(w/v), about 0.6% (w/v), about 0.7% (w/v), about 0.8% (w/v), about 0.9%(w/v), about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v),or about 5% (w/v) of the surfactant.

When using more than one surfactant, the suspension may contain the sameor different amounts of each.

In addition to a surfactant, the suspensions may comprise a stabilizer.In one embodiment, the stabilizer is selected from hydroxy propylcellulose, hydroxypropylmethyl cellulose, hydroxyethylcellulose,polyvinyl, pyrrolidone, carboxymethylcellulose, Pemulen®, and Pemulen®TR-2. Pemulen® is the trade name for high molecular weight, crosslinkedcopolymers of acrylic acid and C10-C30 alkyl acrylate produced byLubrizol Corp. Pemulen® TR-2 is a C10-C30 alkyl acrylate crosspolymercontaining a higher level of hydrophobic groups than other Pemulen®polymers. The vehicle may contain all of these stabilizers, or none ofthem, or it may contain one, two, three, four, or five of them.

One can use between about 0.01% (w/v) and about 10% (w/v) of thestabilizer. In one embodiment, the suspensions contain about 0.01% (w/v)to about 1% (w/v), or about 0.01% (w/v) to about 0.1% (w/v), or about0.1% (w/v) to about 1% (w/v) of the stabilizer. For example, thesuspensions may contain about 0.01% (w/v), about 0.02% (w/v), about0.03% (w/v), about 0.04% (w/v), about 0.05% (w/v), about 0.06% (w/v),about 0.07% (w/v), about 0.08% (w/v), about 0.09% (w/v), about 0.1%(w/v), about 0.2% (w/v), about 0.3% (w/v), about 0.4% (w/v), about 0.5%(w/v), about 0.6% (w/v), about 0.7% (w/v), about 0.8% (w/v), about 0.9%(w/v), about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v),about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9%(w/v), or about 10% of the stabilizer.

When using more than one surfactant, the suspension may contain the sameor different amounts of each.

In addition to a surfactant, the vehicle may also comprise a tonicityadjustor selected from glycerin, mannitol, sodium citrate dihydrate,potassium chloride, boric acid, and sodium borate decahydrate. Thetonicity adjustor is added as needed to achieve the desired tonicity;the vehicle may contain all of these tonicity adjusters, or none ofthem, or it may contain one, two, three, four, or five of them. In oneembodiment, the tonicity adjustors are present in an amount of betweenabout 0.1% (w/v) and about 10% (w/v). When using more than one tonicityadjustor, the suspension may contain the same or different amounts ofeach.

The suspension usually contains water, in an amount sufficient toprovide a desired pH, tonicity, and other characteristics that wouldmake the suspension appropriate for administration to the eye.

Methods of Preparing Suspension of Cyclosporin A Form 2

The formulations of the invention may be made by mixing cyclosporin Aform 2 with the appropriate surfactants, stabilizers, and tonicityadjustors, as described above, to form a suspension If fine particles ofcyclosporin A are desired, the suspension is then milled using a highpressure homogenizer, such as those commercially available fromMicrofluidics Intl Corp. of Newton, Mass. A unique and surprisingproperty of cyclosporin form A 2 is that it may be milled, if desired,to obtain a suspension with an average particle size (d90) of less than1 μm. The cyclosporin A in such a nanosuspension has higherbioavailability compared to other (macro) suspensions of cyclosporin A,due to the higher surface area available for dissolution;bioavailability is further enhanced because the smaller particles enablethe Cyclosporin A to be retained on the eye longer. The smallerparticles of the nanosuspensions result in a formulation with a lowerpotential to produce a foreign body sensation or other irritation that asubject perceives when the formulation is instilled in the eye. Also,because the particles are smaller, they associate more readily withsurfactants and stabilizers, thereby permitting one to use lowerconcentrations of them.

After the cyclosporin A form 2 suspension is milled, it is diluted toobtain the final product.

Methods of Treatment

Compositions of the invention may be used to treat any condition of theeye which is known to be amenable to topical treatment with cyclosporinA (such as with Restasis®) at the concentrations stated here. Forexample, compositions of the invention may be used to treat patientssuffering from dry eye, to treat blepharitis and meibomian glanddisease, to restore corneal sensitivity that has been impaired due torefractive surgery on the eye, to treat allergic conjunctivitis andatopic and vernal keratoconjunctivitis, and to treat pterygia,conjunctival and corneal inflammation, keratoconjunctivitis, graftversus host disease, post-transplant glaucoma, corneal transplants,mycotic keratitis, Thygeson's superficial punctate keratitis, uveitis,and Theodore's superior limbic keratoconjunctivitis, among otherconditions.

The International Dry Eye Workshop (DEWS) defines dry eye as “amultifactoral disease of the tears and ocular surface that results insymptoms of discomfort, visual disturbance, and tear film instabilitywith potential damage to the ocular surface, accompanied by increasedosmolarity of the tear film and inflammation of the ocular surface.” Itincludes those conditions, such as keratoconjunctivitis sicca, that arecaused by tear deficiency or excessive evaporation of tears.

Blepharitis is a chronic disorder producing inflammation of the anteriorand posterior lid margin, with involvement of skin and its relatedstructures (hairs and sebaceous glands), the mucocutaneous junction, andthe meibomian glands. It can also affect the conjunctiva, tear film, andthe corneal surface in advanced stages and may be associated with dryeye. Blepharitis is commonly classified into anterior or posteriorblepharitis, with anterior affecting the lash bearing region of thelids, and posterior primarily affecting the meibomian gland orifices.

Meibomian gland disease most often occurs as one of three forms: primarymeibomitis, secondary meibomitis, and meibomian seborrhea. Meibomianseborrhea is characterized by excessive meibomian secretion in theabsence of inflammation (hypersecretory meibomian gland disease).Primary meibomitis, by contrast, is distinguished by stagnant andinspissated meibomian secretions (obstructive hypersecretory meibomiangland disease). Secondary meibomitis represents a localized inflammatoryresponse in which the meibomian glands are secondarily inflamed in aspotty fashion from an anterior lid margin blepharitis.

Impaired corneal sensitivity often occurs after refractive surgery, suchas photorefractive keratectomy, laser assisted sub-epitheliumkeratomileusis (LASEK), EPI-LASEK, customized transepithelialnon-contact ablation, or other procedures in which the corneal nervesare severed. Impaired corneal sensitivity may also occur after viralinfection, such as by HSV-1, HSV-2, and VZV viruses. Patients withimpaired corneal sensitivity often complain that their eyes feel dry,even though tear production and evaporation may be normal, suggestingthat “dryness” in such patients is actually a form of corneal neuropathythat results when corneal nerves are severed by surgery or inflamedafter viral infection.

Allergic conjunctivitis is an inflammation of the conjunctiva resultingfrom hypersensitivity to one or more allergens. It may be acute,intermittent, or chronic. It occurs seasonally, that is, at only certaintime of the year, or it occurs perennially, that is, chronicallythroughout the year. Symptoms of seasonal and perennial allergicconjunctivitis include, in addition to inflammation of the conjunctiva,lacrimation, tearing, conjunctival vascular dilation, itching, papillaryhyperplasia, chemosis, eyelid edema, and discharge from the eye. Thedischarge may form a crust over the eyes after a night's sleep.

Atopic keratoconjunctivitis is a chronic, severe form of allergicconjunctivitis that often leads to visual impairment. Symptoms includeitching, burning, pain, redness, foreign body sensation, lightsensitivity and blurry vision. There is often a discharge, especially onawakening from a night's sleep; the discharge may be stringy, ropy, andmucoid. The lower conjunctiva is often more prominently affected thanthe upper conjunctiva. The conjunctiva may range from pale, edematous,and featureless to having the characteristics of advanced disease,including papillary hypertrophy, subepithelial fibrosis, fornixforeshortening, trichiasis, entropion, and madarosis. In some patientsthe disease progresses to punctate epithelial erosions, cornealneovascularization, and other features of keratopathy which may impairvision. There is typically goblet cell proliferation in the conjunctiva,epithelial pseudotubular formation, and an increased number ofdegranulating eosinophils and mast cells in the epithelium. CD25+Tlymphocytes, macrophages, and dendritic cells (HLA-DR⁺, HLA-CD1+) aresignificantly elevated in the substantia propria.

Like atopic keratoconjunctivitis, vernal keratoconjunctivitis is asevere form of allergic conjunctivitis, but it tends to affect the upperconjunctiva more prominently than the lower. It occurs in two forms. Inthe palpebral form, square, hard, flattened, closely packed papillae arepresent; in the bulbar (limbal) form, the circumcorneal conjunctivabecomes hypertrophied and grayish. Both forms are often accompanied by amucoid discharge. Corneal epithelium loss may occur, accompanied by painand photophobia, as may central corneal plaques and Trantas' dots.

EXAMPLES

The invention is further illustrated by the following examples.

Example 1

The inventors prepared the following compositions:

TABLE 1 Formulations of cyclosporin A prepared in Example 1 A B C D E FIngredient Concentration % (w/v) CsA 0.01 to 0.05 Polysorbate 80 0.001to 0.05 Glycerin 1 1.2 1.2 1 1.2 2.2 Mannitol 0.5 CMC 0.5 HEC 0.1-0.5Pemulen TR-2 0.01-0.1 0.05-0.1 HPMC 0.1-1.0 PVP 0.1-10.0 Sodium Citrate0.4 0.4 0.4 0.4 0.4 Dihydrate Potassium 0.14 Chloride Boric Acid 0.250.25 0.25 0.25 0.25 Sodium Borate 0.41 0.41 0.41 0.41 0.41 DecahydratePurified Water qs Qs qs qs qs Qs

The formulations of Table 1 were prepared by the following process:

-   -   1. Preparation of Concentrated Cyclosporin A Nanosuspensions        -   a. Cyclosporin A was mixed with an appropriate vehicle to            form a suspension. The concentration of Cyclosporin A in            this suspension is in the range of 1-10%.        -   b. The cyclosporin A suspension was milled using a high            pressure homogenizer (a Microfluidizer®, manufactured by            Microfluidics, Newton, Mass.) or a ball mill to get            nanosuspensions such that d90<11 Jm.        -   c. Vehicles used for preparation of nanosuspension            concentrate are as listed in Table 1.    -   2. Preparation of Final Product        -   a. The concentrated nanosuspensions prepared in Step-1 were            diluted in vehicles suitable for ophthalmic dosing to obtain            final product at required dose strength of cyclosporin A.            Vehicles suitable for dilution may contain buffers,            stabilizers, gelling agents and/or dilution to obtain final            formulations with desired concentration of CsA.        -   b. Compositions of nanosuspension formulations prepared            after dilution of the nanosuspension concentrates are listed            in Table 2.

Particle-size distribution of the different Formulations are shown inFIGS. 1, 2, and 3.

FIG. 1 shows the particle size distribution of cyclosporin A Form 2nanosuspensions prepared using a high pressure homogenizer compared tosuspensions prior to milling.

FIG. 2 compares the particle size distribution of cyclosporin A Form 2nanosuspensions prepared using different surfactants and stabilizers.

FIG. 3 shows the particle size distribution of Formulation diluted invehicle containing Na CMC (Table-2, vehicle A). Note that no change inparticle size is seen in the diluted Formulations over 2 weeks ascompared to the nanosuspension concentrate.

Average particle size of cyclosporin A Form 3 are shown in FIG. 4.Cyclosporin A Form 2 Forms a nanosuspensions (particle size <1 μm) whileForm 3 does not.

These experiments show that Form 2 consistently produced nanosuspensionswith lower particle size than any other crystalline form of CsA. Thesmaller particle size of Form 2 nanosuspensions is an advantage overother forms; among other reasons, it is expected to show higherbioavailability due to larger surface area for dissolution and longerretention in the eye, as well as improved physical stability.

Example 2

The inventors prepared the cyclosporin A nanosuspensions listed in Table2, below:

TABLE 2 Formulations of cyclosporin A prepared in Example 2 Restasis ® AA1 B1 D Ingredient Concentration (% w/w) Nano- CsA — 0.05 0.05 0.01 0.05suspension Polysorbate 80 — 0.005 0.005 0.001 — Na glychoholate — — — —from sus- pension Oil CsA 0.05 — — — — Phase Castor oil 1.25 — — — —Aqueous PS80 1 0.045 0.045 0.049 0.05 Vehicle Glycerin 2.2 2.2 2.2 2.22.2 Citric acid * — — 0.007 — — H2O Na2HPO4 * — — 0.134 — — 7H2O PemulenTR-2 0.05 0.05 — 0.05 0.05 Gellan Gum — — 0.6 — — Purified water QS QSQS QS QS

The inventors administered the above formulations to NZW female rabbits(two rabbits total, one formulation per eye) in a single topical dose.Results are summarized in FIGS. 5-13. They show that formulation B1delivers a 5-fold lower dose of cyclosporin A (0.01% versus 0.05%) butmaintains comparable cornea exposure to 0.05% Restasis® as well asimproves delivery to the bulbar and palpebral conjunctiva.

What is claimed is:
 1. A method of treating a condition selected fromdry eye, blepharitis, meibomian gland disease, impaired cornealsensitivity, allergic conjunctivitis, atopic keratoconjunctivitis,vernal keratoconjunctivitis, and pterygia, the method comprising thestep of administering to a patient having such a condition a formulationcomprising particles of cyclosporin A form 2 and a vehicle, wherein theaverage size (d90) of the particles is less than about 10 μm.
 2. Themethod of claim 1, wherein the average size of the particles is lessthan about 1 μm.
 3. The method of claim 1, wherein the condition is dryeye.